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Biomedical subjects

L D Falo

Publications and source records attributed to L D Falo.

At least 19 recordsLinked to original sources
PubMed2005

'Survival gene' Bcl-xl potentiates DNA-raised antitumor immunity.

T-cell priming is strongly affected by the longevity of antigen-bearing dendritic cells (DCs), which are typically short-lived in lymphoid tissues. 'Survival gene' Bcl-xl is critical for the lifespan of DCs in vivo. Here, we showed that in vivo coadministration of Bcl-xl under control of the DC-specific promoter (CD11c-Bcl-xl) and TRP2hsp70 DNA prolonged T-cell stimulation by DCs and augmented TRP2-specific-IFN-gamma-producing CD8+ T-cell responses. Consistent with these findings, enhanced protection and significant therapeutic immunity to B16 melanoma was generated by this coimmunization strategy, which also augmented therapeutic immunity to GL-26 tumor. In this B16 melanoma model, results from animal experiments with depletion of immune cells indicate that CD8+ T cells and NK cells are important in the antitumor immunity induced by this coimmunization strategy. These observations suggest that 'survival gene' Bcl-xl potentiates the magnitude of antigen-specific-CD8+ T-cell responses and the efficacy of antitumor immunity induced by DNA vaccine, and is relevant for the design of in vivo targeted DC-based vaccine strategies to improve immunity against cancer.

Animals↗
PubMed2001

Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation.

Although it is known that dendritic cells (DCs) produce cytokines, there is little information about how cytokine synthesis is regulated during DC development. A range of cytokine mRNA/proteins was analyzed in immature (CD86-) or mature (CD86+) murine bone marrow (BM)- derived DCs. Highly purified, flow-sorted, immature DCs exhibited higher amounts of interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1), and macrophage migration inhibitory factor (MIF) mRNA/protein than mature DCs. After differentiation, DC up-regulated the levels of IL-6 and IL-15 mRNA/protein and synthesized de novo mRNA/protein for IL-12p35, IL-12p40, and IL-18. Although immature BM-derived DCs did not stimulate naive allogeneic T cells, mature DCs elicited a mixed population of T helper (Th) 1 (mainly) and Th2 cells in 3d-mixed leukocyte reactions. CD86+ BM DCs switched to different cytokine patterns according to whether they were terminally differentiated by lipopolysaccharide (LPS) or CD40 ligation. Although both stimuli increased IL-6, IL-12p40, IL-15, and TNF-alpha mRNA/protein levels, only LPS up-regulated transcription of IL-1alpha, IL-1beta, IL-12p35, and MIF genes. Although LPS and CD40 cross-linking increased the T-cell allostimulatory function of BM DCs, only LPS stimulation shifted the balance of naive Th differentiation to Th1 cells, a mechanism dependent on the up-regulation of IL-12p35 and not of IL-23. These results demonstrate that, depending on the stimuli used to terminally mature BM DCs, DCs synthesize a different pattern of cytokines and exhibit distinct Th cell-driving potential.

Animals↗
PubMed2001

Aspirin inhibits in vitro maturation and in vivo immunostimulatory function of murine myeloid dendritic cells.

Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-kappaB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-kappaB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.

Animals↗
PubMed2001

Regression of human mammary adenocarcinoma by systemic administration of a recombinant gene encoding the hFlex-TRAIL fusion protein.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL, is a new member of the TNF family. It can specifically induce apoptosis in a variety of human tumors. To investigate the possibility of employing the TRAIL gene for systemic cancer therapy, we constructed a recombinant gene encoding the soluble form of the human Flt3L gene (hFlex) at the 5' end and the human TRAIL gene at the 3' end. Such design allows the TRAIL gene product to be secreted into the body circulation. We have also demonstrated that the addition of an isoleucine zipper to the N-terminal of TRAIL greatly enhanced the trimerization of the fusion protein and dramatically increased its anti-tumor activity. The fusion protein reached the level of 16-38 microg/ml in the serum after a single administration of the recombinant gene by hydrodynamic-based gene delivery in mice. A high level of the fusion protein correlated with the regression of a human breast tumor established in SCID mice. No apparent toxicity was observed in the SCID mouse model. In addition, the fusion protein caused an expansion of the dendritic cell population in the C57BL/6 recipient mice, indicating that the hFlex component of the fusion protein was functional. Thus, the hFlex-TRAIL fusion protein may provide a novel approach, with the possible involvement of dendritic cell-mediated anti-cancer immunity, for the treatment of TRAIL-sensitive tumors.

Adenocarcinoma↗
PubMed2001

Dermal-resident CD14+ cells differentiate into Langerhans cells.

Epidermal Langerhans cells (LCs) show extraordinary immunostimulatory capacity and play a key role in the initiation and regulation of immune responses. Studies of LC biology are currently the focus of efforts to engineer immune responses and to better understand the immunopathology of cutaneous diseases. Here we identified and characterized a population of LC precursors that were resident in human skin. These immediate precursors expressed CD14, langerin and functional CCR6. When cultured with transforming growth factor-beta1 alone, they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage-colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells. Identification and characterization of these LC precursors provided insight into LC biology and the mechanism(s) through which LCs repopulate the epidermis.

Antigens↗
PubMed2001

Direct transfection and activation of human cutaneous dendritic cells.

Gene therapy techniques can be important tools for the induction and control of immune responses. Antigen delivery is a critical challenge in vaccine design, and DNA-based immunization offers an attractive method to deliver encoded transgenic protein antigens. In the present study, we used a gene gun to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Our studies demonstrate that when delivered to human skin, gold particles are observed primarily in the epidermis, even when high helium delivery pressures are used. We demonstrate that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates the presence of transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Importantly, transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL. Taken together, our results demonstrate direct transfection, activation, and antigen-specific stimulatory function of in situ transduced human Langerhans cells.

Biolistics↗
PubMed2000

Generating and regulating immune responses through cutaneous gene delivery.

The combination of immunization strategies with gene therapy methods constitutes a powerful tool for the purpose of genetic immunization. The cutaneous microenvironment, rich in professional antigen-presenting cells and accessory cells capable of initiating and controlling the intensity of specific immune responses, makes the skin a unique target for the expression of transgenic antigens. The fact that epidermal and dermal dendritic cells can be directly transfected using genetically engineered vectors allows in vivo manipulation of immune responses by modifying the function of these distinctive antigen-presenting cell populations. Importantly, coexpression of antigenic proteins together with immunostimulatory molecules, and/or adjuvant or leader sequences, makes possible the engineering of antigen-specific immune responses. Even though most of the mechanisms related to DNA immunization remain to be explored, the skin has emerged as an ideal target for evolving genetic vaccination techniques.

Epidermis↗
PubMed2000

Intravenous injection of naked DNA encoding secreted flt3 ligand dramatically increases the number of dendritic cells and natural killer cells in vivo.

The trace number of dendritic cells (DCs) present in tissues has limited the study of DC biology and development of clinical applications utilizing DCs. Here we show that hydrodynamics-based gene delivery of naked DNA encoding secreted human flt3 ligand (hFLex) can dramatically increase the number of functional DCs and natural killer (NK) cells. After a single injection of the hFLex gene, hFLex levels in mouse serum reached approximately 40 microg/ml and remained above 1 microg/ml for 5-6 days. Sustained levels of serum hFLex correlated with significant increases in the size of the lymphoid organs and in the proportion of dendritic cells and NK cells in both lymph nodes and spleen. The increase in DC and NK cell numbers started from day 5, and reached peak levels between day 8 and day 12. The levels then returned to normal on day 20. These DCs and NK cells were functional as evidenced by mixed leukocyte reactions and lysis of YAC-1 cells, respectively. These results suggest that delivery of the hFLex gene provides a simple, efficient, and inexpensive way of increasing DC and NK cell populations in vivo, and may have broad applications in the further study of DC and NK cell biology and in the development of immunotherapy strategies.

Animals↗
PubMed2000

Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines.

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.

Adoptive Transfer↗
PubMed2000

Recombinant adenovirus induces maturation of dendritic cells via an NF-kappaB-dependent pathway.

Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC). Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-kappaB-DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection with rAd-IkappaB, an rAd-encoding the dominant-negative form of IkappaB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.

Adenoviridae↗
PubMed1999

A particulate viral protein vaccine reduces viral load and delays progression to disease in immunized ponies challenged with equine infectious anemia virus.

Immunization regimens that induce a broadly reactive cytolytic T lymphocyte (CTL) response specific for lentiviral antigens have emerged as the leading candidates in efficacy trials conducted in both animal modelshumans. To date, lentivirus vaccination strategies have overlooked one such immunization strategy, namely the use of particulate antigens. To evaluate the efficacy of targeting antigen into the phagocytic pathway to elicit a cell-mediated immune response to lentiviral antigens, we initiated the first study of a particulate-based vaccination protocol using a large animal model system. Gradient-purified equine infectious anemia virus (EIAV) was covalently coupled to glutaraldehyde-activated iron oxide beads. In vitro studies demonstrated the effectiveness of the inactivated whole virus particulate to prime antigen presenting cells for the activationexpansion of virus-specific CD8(+) CTL. The in vivo effectiveness of the particulate antigen was evaluated by experimental immunization of ponies. Ponies receiving the viral particulate vaccinechallenged with infectious EIAV had a delayed progression to diseasea reduced viral load compared with infected ponies that had not been vaccinated. Interestingly, in vitro virus-specific CTL activity was detected in only one of four immunized animals at the day of challenge. The beneficial effects of the particulate vaccine regimen were not clearly associated with any in vitro measurable parameters of the virus-specific cellular or humoral immune responses elicited by the vaccine at the day of challenge. However, within 3 weeks after virus challenge, anamnestic humoral responses characterized by a rapid emergence of neutralizing activity in the seruma predominance of conformationally dependent epitopes recognized by virus-specific antibodies were observed in the vaccinates. Taken together, further studies are clearly warranted in large animal model systems using a particulate-based vaccine regimen considering the beneficial effects of this regimen in our studythe protective effects of particulate antigen delivery in the murine model.

Animals↗
PubMed1998

Physical interaction between dendritic cells and tumor cells results in an immunogen that induces protective and therapeutic tumor rejection.

Dendritic cells (DCs) are potent professional APCs capable of presenting Ag in the context of costimulatory signals necessary for T cell activation. Although tumor cells express target Ags, they are generally incapable of stimulating an immune response. We show that the short term physical interaction of DCs and tumor cells, with or without cell fusion, results in rapid, efficient, and stable DC-tumor cell association. Immunization of naive mice with unselected, irradiated DC-tumor cell conjugates induces tumor-specific CD8+ cytotoxic T cells and protection from lethal tumor challenge. Furthermore, the immunogenicity of this cellular vaccine is dependent on the physical interaction of DCs and tumor cells before injection. Immunization with DCs and tumor cells after physical interaction can result in the regression of established tumors and persistent antitumor immunity. These results suggest that immunization with DC-tumor cell vaccines may be a simple, rapid, and potent strategy for tumor immunotherapy.

Animals↗
PubMed1998

DNA immunization targeting the skin: molecular control of adaptive immunity.

DNA-based immunization represents a novel approach for vaccine development. Recombinant DNA techniques are used to clone DNA sequences encoding antigens of choice into eukaryotic expression plasmids, which are readily and economically amplified in bacteria and recovered with a high degree of purity. For immunization, plasmid DNA is either coated onto microscopic gold particles and bombarded into skin using a gene gun or injected into skin or muscle. Expression of administered genes results in the induction of humoral and cellular immune responses against the encoded antigen. DNA immunization is capable of inducing protective immunity in a number of animal models of infectious disease and cancer. Recent studies suggest that antigen-specific cytotoxic T lymphocyte induction occurs through the presentation of appropriate peptides in the context of major histocompatibility complex molecules on bone marrow-derived professional antigen presenting cells. Following DNA inoculation into the skin, Langerhans cells and/or dermal dendritic cells are believed to acquire the newly synthesized antigen, either through direct transfection or via antigen uptake from transfected keratinocytes, and migrate to regional lymph nodes where they stimulate primary T cell responses. The nature of the immune response depends on the route, method, and timing of DNA delivery and can also be influenced by co-delivery of plasmids encoding immunomodulating cytokines like IFN-alpha, IL-2, or IL-12 and costimulatory molecules like B7-1. While many aspects of the biology of cutaneous DNA immunization remain unknown, the skin appears to offer unique potential as a target for DNA-based immunization.

Animals↗
PubMed1998

Cytotoxic T cells in basal cell carcinomas of skin.

Previous studies have suggested the importance of CD8+ cytotoxic T-cells of hosts against neoplasms. Earlier studies and our previous investigation showed that a majority of tumor infiltrating T-cells in human basal cell carcinomas (BCCs) belonged to CD4+ T-cells. CD8+ cells were also present in the peritumor areas of human BCCs, but in smaller numbers. Published evidence indicates the importance of cytotoxic T-cells in antitumor immunity. Cytotoxic T-cells have been identified by using monoclonal antibodies against various cytotoxic T-cell components. In this study, we used monoclonal antibodies to perforin to evaluate the role of cytotoxic T-cells in the host response against basal cell carcinomas. Perforin-expressing T-cells could be identified in the infiltrate of BCCs in frozen tissue sections, and also in antigen-retrieved paraffin-embedded sections of BCCs, and the presence of perforin-expressing T-cells correlated with the infiltration of CD8+ T-cells. These results suggest that cytotoxic T-cells play a role in host defense against human BCCs.

CD4-CD8 Ratio↗
PubMed1997

Epidermal dendritic cells induce potent antigen-specific CTL-mediated immunity.

Professional antigen-presenting cells (APCs) are required for the initiation of an immune response. Dendritic cells (DCs) are the most potent APCs identified thus far and can present antigen in the context of co-stimulatory signals required for the stimulation of both primed and naïve T cells. Cytotoxic T lymphocytes (CTLs) are critical to the immune response against tumors or virally infected cells. Optimal stimulation of antigen-specific CTLs is the goal of evolving immunization strategies for the prevention or therapy of viral infections and tumors. Epidermal dendritic cells (eDCs), or Langerhans cells, can present antigens for the stimulation of CD4+ T cell dependent anti-tumor immunity and may play a role in tumor surveillance. The capacity of eDCs to induce tumor-specific CD8+ CTL immunity has not been determined. We have previously shown that DCs derived from bone marrow precursors (BmDCs) under the influence of cytokines can induce protective, antigen-specific CTL-mediated anti-tumor immunity. Here we show that subcutaneous immunization with ovalbumin (OVA) peptide (SIINFEKL(257-264))-pulsed eDCs induced OVA-specific, CD8+ CTLs that lyse the OVA-expressing target. Furthermore, mice vaccinated with OVA peptide-pulsed eDCs were completely protected from subsequent challenge by the OVA-expressing melanoma MO5. The capacity of peptide-pulsed eDCs to induce CTL-mediated immunity is directly dependent on the dose of eDCs administered. Importantly, the APC capacity of eDCs is comparable to that of BmDCs, as mice immunized with eDC populations containing at least as many class II+/B7.2+ cells as populations of BmDCs were equally protected against challenge with MO5. These results demonstrate that eDCs can be potent inducers of antigen-specific CD8+ CTL-mediated immunity. They suggest that eDCs may be important targets for antigen delivery strategies aimed at inducing antiviral or anti-tumor immunity.

Adoptive Transfer↗
PubMed1996

Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity.

Cytotoxic T lymphocytes (CTLs) are a critical component of the immune response to tumors. Tumor-derived peptide antigens targeted by CTLs are being defined for several human tumors and are potential immunogens for the induction of specific antitumor immunity. Dendritic cells (DC) are potent antigen-presenting cells (APCs) capable of priming CTL responses in vivo. Here we show that major histocompatibility complex class I-presented peptide antigen pulsed onto dendritic APCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. The immunity is antigen specific, requiring expression of the antigen gene by the tumor target, and is eliminated by in vivo depletion of CD8+ T cells. Furthermore, mice that have rejected the transfected tumor are protected from subsequent challenge with the untransfected parent tumor. These results suggest that immunization strategies using antigen-pulsed DC may be useful for inducing tumor-specific immune responses.

Amino Acid Sequence↗
PubMed1996

Treatment of pyogenic granulomas with the 585 nm pulsed dye laser.

BACKGROUND: Current therapeutic alternatives for pyogenic granulomas include surgical excision, electrodesiccation and curettage, cryotherapy, and ablation with CO2 or continuous-wave vascular lasers. OBJECTIVE: Our purpose was to investigate the use of the 585 nm flashlamp-pumped pulsed dye laser (585 nm PDL) for the treatment of pyogenic granulomas in terms of efficacy, advantages in technique, and side effects. METHODS: Eighteen patients with symptomatic pyogenic granulomas in a variety of locations were treated with the 585 nm PDL and examined. RESULTS: Sixteen of 18 treated patients demonstrated both symptomatic and clinical clearing of the lesions with excellent cosmetic results after treatment. The two patients who dropped out after one to two 585 nm PDL treatments were eventually treated successfully with electrodesiccation and curettage. No postoperative complications and no persistent pigmentary changes or scarring were observed. The procedure required no anesthesia, and postoperative care was limited to the application of a topical antibiotic ointment. CONCLUSION: Our experience suggests that treatment of pyogenic granulomas with the 585 nm PDL is a safe, effective, and reasonable alternative to conventional therapy.

Administration, Cutaneous↗